Mechanism of saikogenin G against major depressive disorder determined by network pharmacology

Many classic decoctions of Chinese medicine including Radix Bupleuri are used to treat major depressive disorder (MDD). Saikosaponin D is a representative bioactive ingredient discovered in Radix Bupleuri. The mechanism of saikogenin G (SGG) as a metabolite in MDD remains unclear to date. This study aims to elucidate the mechanism of SGG in treating MDD with network pharmacology. We evaluated the drug likeness of SGG with SwissADME web tool and predicted its targets using the SwissTargetPrediction and PharmMapper. MDD-related targets were identified from the following databases: DisGeNET, DrugBank, Online Mendelian Inheritance in Man, and GeneCards. The common targets of SGG and MDD were imported to the STRING11.0 database, and then a protein–protein interaction network was constructed. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were analyzed with DAVID 6.8 database. The molecular weight of SGG was 472.7 g/mol, the topological polar surface area was 69.92 A2 <140 A2, the octanol/water partition coefficient (Consensus LogP0/W) was 4.80, the rotatable bond was 1, the hydrogen bond donors was 3, and the hydrogen bond acceptors was 4. A total of 322 targets of SGG were obtained and there were 1724 MDD-related targets. A total of 78 overlapping genes were selected as targets of MDD treatment including albumin, insulin-like growth factor I, mitogen-activated protein kinase 1, proto-oncogene tyrosine-protein kinase Src, and epidermal growth factor receptor. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis suggested that proteoglycans in cancer, pathways in cancer, prostate cancer, hypoxia-inducible factor-1, central carbon metabolism in cancer, estrogen, PI3K-Akt, ErbB, Rap1, and prolactin signaling pathways played an important role(P < .0001). This study showed that SGG exhibits good drug-like properties and elucidated the potential mechanisms of SGG in treating MDD with regulating inflammation, energy metabolism, monoamine neurotransmitters, neuroplasticity, phosphocreatine-creatine kinase circuits, and so on.


Introduction
Depressive disorder is a common mental disorder. Main subcategories of depressive disorders include major depressive disorder (MDD) and dysthymia. [1] About 322 million people in the world are suffering from depression. Recurrent and long-lasting depression is characterized by continuous sadness, lack of interest, sense of guilt, poor attention, and so on. People with depression cannot work and live normally, commit nonsuicidal Medicine cocos (Schw.) Wolf., licorice, Zingiber officinale Roscoe), Sini Power (Radix Bupleuri, Paeoniae Radix Alba, Aurantii Fructus Immaturus, licorice), and Xiaochaihu decoction (Radix Bupleuri, Arum ternatum Thunb., Panax Ginseng C. A. Mey., licorice, Scutellariae radix, Z. officinale Roscoe, Jujubae Fructus) have been used for over thousands of years in China to treat MDD. [4] Saikosaponins (Saikosaponin A, Saikosaponin B, Saikosaponin D, and Saikosaponin K) are representative bioactive ingredients discovered in Radix Bupleuri, a common component of the mentioned decoctions above. Saikosaponin D protects the nervous system, alleviates neurological dysfunction, rescues sexual behavior deficits, improves depression-like behavior, and prevents Alzheimer disease, osteoarthritis, breast cancer, and leukemia. [5][6][7][8][9][10][11] Saikosaponin D as a triterpene saponin glycoside is hydrolyzed to prosaikogenin G and then to saikogenin G (SGG), which is subsequently absorbed into the circulatory system. [12] Previous studies focused on the biological effects of saikosaponin D. However, the bioactivity and mechanism of SGG in MDD remain unclear to date.
In the present study, we aimed to elucidate the mechanism of SGG in treating MDD with network pharmacology. We evaluated the druglikeness of SGG and predicted its targets against MDD. A protein-protein interaction (PPI) network was constructed with the common targets. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed with the database. This study provided a thorough investigation and comprehensive analysis of SGG in treating MDD.

PPI network analysis
A PPI network was predicted based on the STRING 11.0 platform (https://string-db.org/). The targets of SGG against MDD were imported to the STRING11.0 database, and then target interaction information was combined and analyzed with Cytoscape software (version 3.7.1; https://www.cytoscape. org/).

GO and KEGG pathway enrichment analyses
Target genes of SGG against MDD were mapped into the Functional Annotation Tool DAVID 6.8 database (https:// david.ncifcrf.gov/) for GO analysis, including the biological process (BP), cell component, and molecular function, and KEGG analysis. A heatmap was plotted by http://www.bioinformatics.com.cn, an online platform for data analysis and visualization.

ADME properties of SGG
The chemical structure of SGG was obtained from the PubChem database and is shown in Figure 1A. In accordance with the RO5, SwissADME prediction showed that the molecular weight of SGG was 472.7 g/mol, which should be <500 g/mol, the topological polar surface area was 69.92 A 2 < 140 A 2 , the octanol/ water partition coefficient (Consensus LogP 0/W ) was 4.80 <5, the rotatable bond was 1 <10, the hydrogen bond donors was 3 no more than 5, and the hydrogen bond acceptors was 4 no more than 10. [19] Results indicated that the properties of SGG were in line with the RO5, and GI absorption was high, suggesting that it had good drug-like properties ( Table 1).

Screening of targets of the SGG in MDD
Based on structure, potential targets of SGG were predicted via the SwissTargetPrediction and PharmMapper databases. In the SwissTargetPrediction database, targets with probability >0 were selected as potential targets (Fig. 1B, C). A total of 322 targets were obtained, including 27 duplicated targets (SwissTargetPrediction (Probability > 0) 67, PharmMapper 282).
After removing redundant data, 1724 target genes relevant to MDD were obtained from the DisGeNET, DrugBank, Online Mendelian Inheritance in Man, and GeneCards databases. Then, a Venn diagram was used to outline 3 common targets in those databases. From an intersection of SGG compound targets and MDD targets, 78 overlapping genes were selected as targets of MDD treatment (Fig. 2).

PPI network analysis
The PPI network of those 78 targets was set up with the STRING database. A total of 76 nodes and 646 edges were exhibited, and the average node degree was 16.6 with a medium confidence score of 0.400. The "Analysis network" tool in Cytoscape software was used to analyze the results and obtain the relevant parameters further (Fig. 3A). We selected key targets on the basis of the 3 parameters of "Degree," "BetweennessCentrality," and "ClosenessCentrality" above the median values of them. The threshold values were degree ≥ 20.5, ClosenessCentrality ≥ 0.547445, and betweenness ≥ 0.006381, and 22 hub nodes were obtained (Fig. 3B).

GO and KEGG pathway enrichment analysis
After using the DAVID 6.8 for enrichment analysis, we obtained 84 KEGG pathways and 296 GO biological processes (P < .05; Table 2). The first 10 results from the GO and KEGG pathway enrichment analyses were selected considering their P values.
As shown in Figure 4A, the top 10 significantly enriched BP terms were protein autophosphorylation, phosphatidylinositol-mediated signaling, peptidyl-tyrosine phosphorylation, positive regulation of cell migration, regulation of phosphatidylinositol 3-kinase signaling, positive regulation of mitogen-activated protein kinase cascade, leukocyte migration, positive regulation of extracellular signal-regulated kinase (ERK)1 and www.md-journal.com ERK2 cascade, positive regulation of smooth muscle cell proliferation, and ERBB2 signaling pathway.
The top 10 enriched cell component terms were caveola, receptor complex, cytosol, extracellular region, extracellular space, perinuclear region of cytoplasm, RNA polymerase II transcription factor complex, mitochondrion, plasma membrane, and extracellular exosome. The top 10 enriched molecular function terms included protein tyrosine kinase activity, steroid hormone receptor activity, insulin receptor substrate binding, phosphatidylinositol-4,5-bisphosphate 3-kinase activity, transmembrane receptor protein tyrosine kinase activity, receptor signaling protein tyrosine kinase activity, steroid binding, identical protein binding, serine-type endopeptidase activity, and heme binding.

Discussion
Major depressive disorder is a multifactorial mental disorder caused by the interplay between environmental factors and genetic susceptibility. [20] Disorders in heredity, neurological hormone, immune function, bioenergetics, mitochondrial oxidative stress, vasomotor factors and free radicals, gender, and developmental factors are associated with MDD. [21][22][23] Existing drugs are not completely effective because of the complex etiology of MDD. Traditional Chinese Medicine has a long history and few side effects in the treatment of MDD. Thus, finding new drugs from Chinese herbal medicine is important.  Gene ontology enrichment analysis showed the complex and comprehensive effects of SGG and revealed several BP terms, such as autophosphorylation, phosphorylation, ERK1 and ERK2 cascade, and mitogen-activated protein kinase cascade. KEGG pathway analysis was performed to reveal the underlying mechanism of antidepressive with SGG, including the HIF-1, estrogen, PI3K-Akt, and ErbB signaling pathways.
Hypoxia-inducible factor-1 is an important transcriptional factor responding to hypoxia, a heterodimer composed of HIF-1α (regulatory subunit) and HIF-1β (constitutive subunit). HIF-1 signaling pathways regulate various BPs, including aging, proliferation and apoptosis, anaerobic metabolism, TCA cycle metabolism, oxygen consumption, inflammation, and angiogenesis. [24,25] Previous studies suggested that a close functional interaction exists between HIF-1 and glucocorticoid receptor (major part of stress response). [26] Li et al [27] found that inhibitor of prolyl hydroxylase (FG-4592) reverses depression-like behavior through the activation of the HIF-1 signaling pathway. Kang et al [22] assumed that elevated levels of HIF-1 activate the phosphocreatine-creatine kinase circuits and regularize the brain energy metabolism as a result of antidepressants via normalization of structural and functional alterations. Insulin, interleukin (IL)-1β, and epidermal growth factor induce the expression of HIF-1α by activating PI3K-Akt signaling pathways. In addition, a previous report revealed that the overexpression of HIF-1α upregulates the expression of inflammatory factors (IL-13, IL-9, IL-1, and tumor necrosis factor-α) by activating the epidermal growth factor receptor/PI3K/AKT pathway. [28] PI3K-Akt signaling plays an important role in many diseases, including cancer, diabetes, obesity, cardiovascular diseases, and neurological diseases (depression, anxiety). [29][30][31] Cao et al [23] reported that icariin significantly ameliorates the seeming symptoms in rats with perimenopausal depression depending on the regulation of PI3K-Akt signaling. Insulin-like growth factor 1, creatine, schisandrin, and liquiritigenin exert an antidepressant effect on animals with depression induced by chronic stress or corticosterone through the PI3K-Akt pathway. [29][30][31][32] Akt (a target of PI3K) acts as a pivot position in the treatment of depression through connecting with downstream targets, including glycogen synthase kinase-3 beta (GSK3β), mammalian target of rapamycin, IκB kinaseα, γ-aminobutyric acid A receptor, and forkhead box O, which are involved in the regulation of glucose and lipid metabolism, synaptic plasticity, angiogenesis, proliferation and apoptosis, synaptic signaling, and mitochondrial function. Moreover, PI3K and AKT activate immune cells by regulating key inflammatory cytokines (IL-8, IL-10, IL-12, IL-23, interferon-α, and interferon-β) and are related to the action of dopamine and serotonin. [33,34] Depression has been associated with increased activity of the immune system and the release of proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α), which are associated with hypothalamic-pituitary-adrenal axis abnormalities in clinical and translational studies. [35,36] Moreover, recent studies have focused on neuroimmune processes   because they may involve the pathophysiology of MDD directly and indirectly through some neurobiological processes related to neuroplasticity-related systems, monoamine system, and the hypothalamic-pituitary-adrenal axis. [37] GSK3β as a downstream target of AKT is crucial for the function of the central nervous system in humans. Phosphorylation on Tyr216 and Ser9 of GSK3β represents positive and negative activities, respectively. [38] Extracellular factors serve many functions, such as regulating the PI3K/AKT pathway and then inhibiting GSK3β by phosphorylation of Ser9. [38] Studies using animal models have indicated that inhibiting the activation of Akt-GSK3β signaling impairs the survival and synaptic plasticity of hippocampal neurons and induces depressive behavior. [39] Further study found that resveratrol may show antidepressant effect by activating the Akt/GSK3β signaling pathway. [35] Phosphatase-mediated phosphorylation plays an important role in the processes of most signaling pathways mentioned above. These situations are consistent with our results that the enriched BP terms are protein autophosphorylation, phosphatidylinositol-mediated signaling, and peptidyl-tyrosine phosphorylation.
The deficiency of central monoamine neurotransmitters dopamine (DA) and serotonin is closely related to depression. At present, selective serotonin reuptake inhibitors, such as fluoxetine, paroxetine, sertraline, fluvoxamine, and citalopram, are widely used in the treatment of depression. Previous studies have shown that the ErbB signaling pathway plays a critical role in depression. [40][41][42] Four closely related ErbB subtypes, namely, ErbB1, ErbB2, ErbB3, and ErbB4, have been identified in humans. Several researchers have reported that NRG1/ErbB signaling plays an important role in the modulation of the midbrain DA system. [40,[43][44][45][46][47] A recent study has confirmed that ketamine exhibits antidepressant effects in adult mice depending on NRG1/ErbB4 signaling. [41] In addition, ErbBs can activate the PI3K pathway directly or indirectly.
Tadeus Reichstein, Adolf Butenandt, and Edward Adelbert Doisy were the first to isolate and characterize estrogen in 1929. [48] Estrogen, including estrone, estradiol, estriol, and estetrol, is a steroid hormone. Estrogen is involved in various physiological processes, including breast and sexual organ development, menstrual cycle, reproduction, cardiovascular protection, bone integrity, cognitive, neuroprotective effects, and antiinflammatory effects, by interacting with estrogen receptors. [39,49] Women show a higher incidence and prevalence of MDD than men. The occurrence of depression increases during the menopause and postpartum periods when the level of estrogen decreases significantly. Several studies have demonstrated that estrogen performs favorable effects in MDD via modulating monoamine neurotransmitters (DA and 5-hydroxytryptamine), inflammatory processes, and dendritic spine number. [50,51] The activation of ERK and PI3K elevates after acute treatment with 17β-estradiol. Estrogen is an important adjunct to antidepressant drug treatments clinically.

(Continued)
The HIF-1, Estrogen, PI3K-Akt, and ErbB signaling pathways are interrelated and interact with each other. We identified key proteins targeted by SGG in the pathways and GO enrichment above and show the related biomechanism in Figure 5.

Conclusion
This study elucidated the potential mechanisms of SGG in treating MDD. Bioinformatics analysis showed that SGG exhibits good drug-like properties, with 78 genes selected as targets of SGG in MDD treatment. Our study also suggested that HIF-1, estrogen, PI3K-Akt, and ErbB signaling pathways regulate inflammation, energy metabolism, dendritic spine number, phosphocreatine-creatine kinase circuits, and BPs in the treatment. This study provided valuable information to investigate the comprehensive and underground effect of SGG in MDD treatment.